In the mitochondrial oxidative phosphorylation system, the energy from the oxidative reactions is used for the generation of a H plus-gradient and associated membrane potential which subsequently drives the phosphorylation of ADP by Pi. Pi-ATP exchange, which is apart of the ADP phosphorylation reaction, is catalyzed by the vesicular energy transducing ATPase complex. Recent work from the laboratory has led to the identification of coupling factor B, a 15,000 dalton -SH containing protein, as a component of the Pi-ATP exchange activity catalyzed by the ATPase complex. The factor is not needed for the oligomycin sensitive ATPase activity. We propose to determine the location of the factor in the membrane, whether it is on the inner or outer surface of the inner mitochondrial membrane. Secondly, we will examine the effect of Factor B in depleted submitochondrial particles of the H plus-uptake associated with ATP breakdown and subsequent relaxation of the gradient due to leakage. A third aim is to incorporate the membrane protein fraction (Fo) derived from the ATPase complex into liposomes and study H plus-transport driven by a K plus-gradient in the presence of valinomycin. Similar experiments will be carried out with Factor B-depleted and supplemented Fo to explore the role of Factor B in the H plus-pumping reaction.